定向进化和半理性设计改造顺式环氧琥珀酸水解酶

Modification of cis-epoxysuccinic acid hydrolase by random mutation combined with semi-rational design

【背景】D(-)-酒石酸是非天然有机酸,在保健品、食品和肿瘤药物合成等行业具有重大应用潜力,目前主要通过生物转化法生产,即顺式环氧琥珀酸水解酶[cis-epoxysuccinic acid hydrolase,CESH(D)]水解顺式环氧琥珀酸(cis-epoxysuccinic acid,ESH)生成D(-)-酒石酸。该法简单温和,但存在CESH(D)酶活转化效率低下的瓶颈问题。【目的】通过基因工程改造,提高CESH(D)的酶活力、温度和pH稳定性。【方法】利用定向进化和半理性设计体外改造CESH(D),高通量筛选出正向突变体;然后对其进行酶学性质研究,包括比酶活、温度和pH对酶催化效率的影响、酶的温度稳定性、pH稳定性及酶促动力学分析;最后通过分子对接等手段分析突变位点影响催化活性的初步机制。【结果】筛选获得4个正向突变体L231P/N226S、V77I、D183E和T223S。与野生型相比,4个突变体的比酶活分别提高2.2、1.6、1.5和1.4倍。其中,L231P/N226S的温度稳定性和pH稳定性较野生型均有显著提高,55℃时催化活性为野生型的1.6倍,pH 6.0时催化活性为野生型的1.2倍。动力学分析发现,突变体L231P/N226S和T223S对底物ESH的亲和力有明显提升,Km值分别为20 mmol/L和21 mmol/L,较野生型分别降低18%和16%。分子对接研究表明,突变位点主要通过改变底物结合口袋促进酶与底物的相互作用,从而影响酶活。【结论】获得了温度和pH稳定性好、催化活性明显提高的CESH(D)突变体,初步分析突变位点影响酶活的机制,为进一步研究CESH(D)的结构-功能关系及深入改造提供指导。

[Background]D(−)-tartaric acid is a non-natural organic acid,which has great application potential in health care products,food,and tumor drug synthesis.At present,D(-)-tartaric acid is produced from cis-epoxysuccinic acid(ESH)hydrolyzed by cis-epoxysuccinic acid hydrolase[CESH(D)]through biotransformation.This method is simple and mild but has bottleneck problems such as low enzyme conversion efficiency of CESH(D).[Objective]To improve the enzyme activity,temperature,and pH stability of CESH(D)through genetic engineering.[Methods]Directional evolution and semi-rational design were used to modify CESH(D)in vitro to screen out forward mutants with high throughput.Then the enzymatic properties were studied,including the enzyme activity,the influence of temperature and pH on the catalytic efficiency of the enzyme,the temperature and pH stability of the enzyme,and enzymatic kinetics analysis.Finally,the primary mechanism of mutation sites affecting catalytic activity was analyzed using molecular docking.[Results]Four positive mutants L231P/N226S,V77I,D183E,and T223S were screened out.The specific enzyme activity of the four mutants was increased by 2.2,1.6,1.5,and 1.4 folds of the wild-type enzyme,respectively.The temperature stability and pH stability of L231P/N226S mutant were significantly higher as compared with the wild-type CESH(D),and its catalytic activity was 1.6 folds at 55℃,and 1.2 folds at pH 6.0 as compared with the wild-type enzyme,respectively.Kinetic analysis showed that the affinity of L231P/N226S mutant and T223S mutant to ESH substrates was significantly increased,with Km values of 20 mmol/L and 21 mmol/L,respectively,which were 18%and 16%lower than that of the wild-type enzyme,respectively.Finally,the results of molecular docking showed that the mutated site promoted the interaction between enzyme and substrate mainly by changing the substrate binding pocket,thus affecting enzyme activity.[Conclusion]Through experiments,CESH(D)mutants with good temperature and pH stability and significantly improved catalytic activity were obtained.The mechanism of mutation site affecting enzyme activity is preliminarily analyzed in this research,which lays a foundation to study the relationship between the structure and function of CESH(D)and its further improvement.